DNA sequences containing a conjugative transfer mechanism

ABSTRACT

The invention relates to a DNA sequence capable of being transferred by conjugation, which comprises the sequence SEQ ID No: 2. The invention further relates to the use of this DNA sequence and to a method of carrying it out.

This application is a 371 of PCT/FR99/03297, filed Dec. 12, 1999.

The present invention relates to novel DNA sequences capable of beingtransferred by conjugation, to plasmids containing these sequences, tobacteria containing these DNA sequences or these plasmids, and to theuse of these bacteria.

Lactic acid bacteria are involved in the production and preservation ofa large number of food products, such as cheese, butter, yogurt, sausageor sauerkraut, among which dairy products are of particular importance.The transformation of milk by lactic acid bacteria is being carried outin ever-larger vats. An understanding of the mechanisms of transfer ofgenetic material is essential for improving the strains of lactic acidbacteria used in these fermentations. The different mechanisms oftransfer of genetic material are transformation, transduction,protoplast fusion and conjugation. Transformation consists in causinggenetic material to enter a bacterium by natural competence, conversionof the cells to protoplasts or electropermeation of the cells.

Transduction consists in causing genetic material to enter a bacteriumby means of a bacteriophage vector.

Protoplast fusion consists in converting two types of cells toprotoplasts so that, after contact, genetic material passes from onestrain into the other strain.

Conjugation consists in bringing two types of cells into contact so thatgenetic material passes from one strain into the other strain by virtueof natural conjugative genes.

Two distinct cases are possible: either the plasmid possesses all thegenes involved in conjugation and said plasmid itself passes into thereceptor strain, or the plasmid possesses only the genetic componentssufficient for its transfer, the other components being present e.g. onanother plasmid (Mobilization of the relaxable Staphylococcus aureusplasmid pC221 by the conjugative plasmid pG01 involves three pC221 loci,S. J. Projan and G. L. Archer, J. Bacteriol. 1989; 171: 1841-1845). Thelatter situation has an advantage: if the plasmid is transferred to areceptor strain without the conjugative plasmid, it will be unable to betransferred again, its dissemination thereby being prevented. Thistechnique of conjugative plasmid transfer also has the advantage ofintroducing genetic material into strains in which this is difficultusing the transfer techniques described above.

A few studies have already made it possible to demonstrate conjugativesystems in lactic acid bacteria:

Genetic analysis of regions of the Lactococcus lactis subsp. lactisplasmid pRS01 involved in conjugative transfer, D. A. Mills, C. K. Choi,G. M. Dunny and L. L. McKay, Applied and Environ. Microbiol. 1994; 60(12): 4413-4420;

Splicing of a group II intron involved in the conjugative transfer ofpRS01 in lactococci, D. A. Mills, L. L. McKay and G. M. Dunny, J.Bacteriol. 1996; 178 (12): 3531-3538.

In the present patent application, a DNA sequence is described whichcomprises at least one conjugative transfer mechanism; this DNA sequencecomprises a functional part of 5333 bp present in the strain Lactococcuslactis FL877 deposited on Sep. 30, 1998 in the CNCM (CollectionNationale de Cultures de Microorganismes) under no. I-2082.

This DNA sequence of 5333 bp (SEQ ID No: 1) was isolated from theplasmids contained in the strain FL877.

More precisely, the fragment of 5333 base pairs (bp) was isolated bytotal digestion, with the restriction enzyme EcoRI, of the plasmidscontained in the strain Lactococcus lactis FL877. This fragment carriesone or more conjugative transfer mechanisms and is capable of beingtransferred to another strain, especially another strain of L. lactis,for example from the strain MG1363 (GASSON M. J., J. Bacteriol. 1983;154: 1-9). This fragment also carries a system of functional replicationin L. lactis.

From the nucleotide sequence SEQ ID No: 1, the Applicant then isolated aDNA sequence of 2590 bp, which alone confers on a plasmid the propertyof being transferred to another strain by conjugation, especially fromthe strain MG1363.

This sequence of 2590 bp (SEQ ID No: 2) can be obtained by the PCR(polymerase chain reaction) method with the aid of appropriateoligonucleotides.

Thus, according to a first feature, the present invention relates to anucleic acid sequence capable of being transferred by conjugation, whichcomprises the sequence SEQ ID No: 2, its complementary strand or anysequence derived from said sequence or from its complementary strand byvirtue of the degeneracy of the genetic code.

The invention preferably relates to a nucleic acid sequence capable ofbeing transferred by conjugation, said sequence being selected from:

a) the nucleotide sequence of 5333 bp (SEQ ID No: 1) or itscomplementary strand;

b) any sequence hybridizing with the sequence a) under strictconditions; or

c) sequences derived from the sequences a) and b) by virtue of thedegeneracy of the genetic code.

The invention relates more particularly to a nucleic acid sequenceselected from:

a) the nucleotide sequence of 2590 bp (SEQ ID No: 2) or itscomplementary strand;

b) any sequence hybridizing with the sequence a) under strictconditions; or

c) sequences derived from the sequences a) and b) by virtue of thedegeneracy of the genetic code.

According to the present invention, “hybridizing under strictconditions” is understood as meaning hybridization under the followingstringency conditions: 42° C. in a 20 mM sodium phosphate buffer (pH6.5) containing 50% of formamide, 5×SSC, 1×Denhardt's, 0.1% of SDS and100 μg/ml of RNA, and then washing at 60° C. in a buffer containing0.1×SSC and 0.1% of SDS.

The invention further relates to DNA sequences which have a high degreeof homology with the above DNA sequences. A high degree of homologymeans a homology (ratio of the identical nucleotides to the total numberof nucleotides) of at least 70%, preferably of at least 80% andparticularly preferably of at least 90% of the nucleotide sequences whenthey are aligned according to the maximum homology by the optimumsequence alignment method of Needleman and Wunsch, J. Mol. Biol. 1970;48: 443-453. This method is used especially in the UWGCG software of theUniversity of Wisconsin: Devereux et al., Nucl. Ac. Res. 1984; 12:8711-8721-option GAP.

The invention further relates to plasmids transformed with one of theDNA sequences according to the invention. These plasmids can be e.g.plasmid pLDP1 (PREVOTS F. et al., FEMS Microbiol. Lett. 1996; 142:295-299) and plasmid pLAB510 derived from pPF107-3. (PREVOTS F. et al.,FEMS Microbiol. Lett. 1998; 159: 331-336), into which one of the DNAsequences according to the invention has been cloned by the conventionaltechniques well known to those skilled in the art.

The invention further relates to bacteria, especially lactic acidbacteria, preferably belonging to the species Lactococcus lactis, whichcontain at least one DNA sequence or one plasmid as defined above.

These bacteria can be used for the conjugative transfer of geneticmaterial, especially genetic material of industrial interest, to astrain of industrial interest. The conjugative transfer mechanism can becarried by a plasmid or by another part of the bacterial genome.

In particular, these bacteria can be used for the conjugative transferof properties such as phage resistance, the ability to ferment lactose,proteolysis, peptidolysis and bacteriocin production, and of genescoding for proteins of pharmaceutical interest, to strains of industrialinterest, particularly in the dairy industry, but also in thepharmaceutical industry.

The strains of industrial interest which can advantageously receivegenetic material with the aid of the DNA sequences according to theinvention, or a plasmid containing them, are e.g. the strains L. lactisssp lactis and L. lactis ssp cremoris.

The invention therefore also relates to these strains of industrialinterest into which said genetic material has been integrated.

The invention will be understood more clearly with the aid of theExamples below, which include experimental results and a discussionthereof Some of these Examples relate to experiments performed for thepurpose of carrying out the invention, while others are Examples ofimplementation of the invention, which are of course given purely by wayof illustration.

A large part of all the techniques described in these Examples, which iswell known to those skilled in the art, is described in detail in thework by Sambrook, Fritsch and Maniatis entitled: “Molecular Cloning; alaboratory manual”, published in 1989 by Cold Spring Harbor Press, NewYork (2nd edition).

The following description will be understood more clearly with the aidof FIGS. 1 and 2 below, which show:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Subcloning of internal fragments of plasmid pLAB500

The following abbreviations are used in this Figure:

Rep+ or −: fragment replicative (+) or non-replicative (−) in MG1363

Rel+ or −: relaxation (+) or non-relaxation (−) in MG1363

Mob+ or −: high efficacy (+) or low efficacy (−) of transfer of theplasmid from MG1363 to LM2301

In addition, the numbers above the sequences represent the base positionon plasmid pLAB500.

FIG. 2: Kinetics of transfer of plasmid pLAB500

EXAMPLE 1 Sequence of the 5333 bp Fragment

The PCR (polymerase chain reaction) technique, described e.g. in thework by Maniatis cited above, makes it possible to amplify a DNAfragment located between 2 suitably chosen oligonucleotides. Thisamplified DNA can easily be ligated to itself if restriction sites areprovided by the oligonucleotides. In fact, the sequences of theseoligonucleotides can contain, at their 5′ end, a heterologous part ofthe DNA to be amplified, consisting e.g. of 10 to 12 base pairs, six ofwhich constitute a restriction site.

The following oligonucleotides were used to construct an erythromycinresistance gene flanked on either side by an EcoRI restriction site:

oligonucleotide 1:

TACATACGCGTCTCATATATACTTTAGATTG (SEQ ID No: 3)

oligonucleotide 2:

TACATACGCGTGACTTAGAAGCAAACTTAAG (SEQ ID No: 4)

oligonucleotide 3:

TTAAATGATCAGAGCTCCACCGCGGTGGCGG (SEQ ID No: 5)

oligonucleotide 4:

TATTTTGATCAGAACAAAAGCTGGGTACCGG (SEQ ID No: 6)

oligonucleotide 5:

ATTTATGATCATTTCCAGTCGGGAAACCTGT (SEQ ID No: 7)

oligonucleotide 6:

AATTTTGATCAAGTATACCTAATAATTTATC (SEQ ID No: 8)

oligonucleotide 7:

TATGTGAATTCGACTTAGAAGCAAACTTAAG (SEQ ID No: 9)

oligonucleotide 8:

TATGTGAATTCAGTATACCTAATAATTTATC (SEQ ID No: 10)

Using oligonucleotides 5 and 1, a 1.1 kb fragment containing the originof replication could be amplified by PCR from plasmid pRC1 (LE BOURGEOISP. et al., Gene 1992; 111: 109-114).

Using oligonucleotides 2 and 6, a 1 kb fragment containing theerythromycin resistance gene could be amplified by PCR from plasmidpRC1.

These two fragments were digested with BclI and MluI and ligated andwere then used to transform E. coli TG1 (GIBSON T. J., Studies on theEpstein-Barr genome, PhD thesis, Cambridge University, Cambridge, UK,1984) to give plasmid pRC1int.

Using oligonucleotides 3 and 4, a 0.3 kb fragment containing severalunique restriction sites could be amplified by PCR from plasmid pRC1.This fragment was digested with BclI and then ligated to plasmidpRC1int, itself digested with BclI. This novel plasmid, containing theerythromycin resistance gene, an origin of functional replication in E.coli but not in L. lactis, and several unique restriction sites, wascalled pRC1N.

Using oligonucleotides 7 and 8, a 939 bp fragment containing theerythromycin resistance gene was amplified by PCR from plasmid pRC1N.This fragment was subsequently digested with EcoRI and then ligated toall the plasmids of the strain L. lactis FL877, which had previouslybeen extracted and digested with the restriction enzyme EcoRI. Thestrain MG1363 was transformed by this ligation mixture. All theresulting erythromycin resistant clones were used as the donor strain ina conjugation experiment (PREVOTS F. et al., FEMS Microbiol. Lett. 1994;117: 7-14) with the receptor strain LM2301 (WALSH P. M. et al., J.Bacteriol. 1981; 146: 937-944), which is resistant to streptomycin andsensitive to erythromycin. The transconjugants are selected for theirresistance to these two antibiotics. The plasmids which had beentransferred to this strain LM2301 by conjugation were extracted and usedto retransform the strain MG1363. Plasmid pLAB500 of 6.3 kb was isolatedin this way; it was capable of replicating in both the strains LM2301and MG1363 and capable of being transferred from the strain MG1363 tothe strain LM2301 with a high efficiency.

After digestion with the restriction enzyme EcoRI, this plasmid gives 2fragments of 1 kb and 5.3 kb. The first fragment contains theerythromycin resistance gene. The last fragment of 5333 bp was entirelysequenced by the method of Sanger et al. (PNAS-USA 1977; 14: 5463).

Analysis of the sequence showed that the 5333 bp fragment possesses 5complete open reading frames of more than 150 bp (FIG. 1).

EXAMPLE 2 Reduction of the Size of Plasmid pLAB500

The PCR technique was applied in order to determine which of the openreading frames are involved in conjugation and which are involved inreplication of the plasmid.

9 oligonucleotides, each comprising an EcoRI or HindIII restrictionsite, were synthesized for this purpose. These oligonucleotides have thefollowing sequences:

oligonucleotide 9:

TATAAGAATTCCGCTCGTGTCGTCGCACC (SEQ ID No: 11)

oligonucleotide 10:

TATAAGAATTCCATAAATCTACTCTATGC (SEQ ID No: 12)

oligonucleotide 11:

TATAAGAATTCGTAGATGAGATTTTAAAGC (SEQ ID No: 13)

oligonucleotide 12:

TATTAGAATTCTATTTAGGGGTATGTAAC (SEQ ID No: 14)

oligonucleotide 13:

TATTAGAATTCCGTGATTTCTTTAGTGCTGTC (SEQ ID No: 15)

oligonucleotide 14:

TATATAAGCTTAGTATACCTAATAATTTATC (SEQ ID No: 16)

oligonucleotide 15:

TATATAAGCTTCACTTCGCAAAATTCGCG (SEQ ID No: 17)

oligonucleotide 16:

TATATAAGCTTGAGGGTCGAGCTTGAGCG (SEQ ID No: 18)

oligonucleotide 17:

TATATAAGCTTCACTCTTTATATGCTAATAC (SEQ ID No: 19)

Using oligonucleotides 14 and 15, a DNA fragment of 2846 bp, containingthe erythromycin resistance gene and ORF E in the form of aHindIII-HindIII fragment, could be amplified by virtue of therestriction sites provided by the oligonucleotides, enabling thisfragment to be ligated to itself.

Likewise, a DNA fragment of 4808 bp, containing the erythromycin geneand ORF B, C, D and E, could be amplified using oligonucleotides 14 and16.

Finally, a DNA fragment of 5169 bp, containing the erythromycin gene andORF B, C, D and E, could be amplified using oligonucleotides 14 and 17.

These DNA fragments were amplified by PCR from a preparation of plasmidpLAB500 with the enzyme ELONGASE® (BRL).

The PCR products were purified by extraction with phenol/chloroform,precipitated with ethanol, digested with the restriction enzyme HindIIIand ligated to themselves. Cloning of these fragments onto themselvesyielded plasmids pLAB501, pLAB502 and pLAB503. These plasmids wereconstructed in the strain MG1363. Their ability to replicate in thisstrain indicates that ORF E codes for a protein involved in replicationof the plasmid. Moreover, over 230 amino acids, this protein shows ahomology of 21.7% with the protein RepE involved in replication of the Ffactor of E. coli.

These plasmids were then tested for their ability to be transferred tothe strain LM2301 by conjugation. Plasmids pLAB502 and pLAB503 have acomparable transfer efficiency to that of pLDP1, the negative control.It can therefore be said that these plasmids do not possess all the DNAnecessary for their conjugative transfer. On the other hand, pLAB501 hasa comparable conjugative transfer efficiency to that of pLAB500, so allthe DNA necessary for the conjugative transfer of pLAB500 must bepresent in pLAB501.

EXAMPLE 3 Subcloning of pLAB500 fragments into plasmid pLDP1

A fragment containing ORF C and D in the form of an EcoRI-EcoRI fragmentof 2068 bp could be amplified by PCR using oligonucleotides 9 and 12.

A fragment containing ORF C in the form of an EcoRI-EcoRI fragment of1201 bp could be amplified by PCR using oligonucleotides 9 and 13.

A fragment containing ORF B and C in the form of an EcoRI-EcoRI fragmentof 2136 bp could be amplified by PCR using oligonucleotides 11 and 13.

A fragment containing ORF B, C and D in the form of an EcoRI-EcoRIfragment of 2602 bp could be amplified by PCR using oligonucleotides 10and 12.

A fragment containing ORF B and C in the form of an EcoRI-EcoRI fragmentof 1735 bp could be amplified by PCR using oligonucleotides 10 and 13.

After digestion with the enzyme EcoRI, each of these fragments wascloned into pLDP1 to give plasmids pLAB504, pLAB505, pLAB506, pLAB507and pLAB508 respectively. Among these plasmids, only pLAB507 wastransferred with a significant efficiency, which was better than that ofthe other plasmids but nevertheless not as good as that of pLAB500. Itcan therefore be said that ORF B, C and D are necessary for conjugativetransfer.

EXAMPLE 4 Subcloning of a pLAB500 Fragment into pLAB510

The following oligonucleotides were used in this subcloning:

oligonucleotide 18:

AGGTTTCCCGACTGGAAATG (SEQ ID No: 20)

oligonucleotide 19:

TACGTGAATTCAGTTTTAAATCAATCTAAAG (SEQ ID No: 21)

oligonucleotide 20:

TACGTGGATCCATCGGCATAATCGTTAAAAC (SEQ ID No: 22)

oligonucleotide 21:

TACGTGAATTCAGAAGAACCCTTAACTAAAC (SEQ ID No: 23)

Plasmid pLDP1 is transferred by conjugation from MG1363 to LM2301 with alow but non-zero efficiency. To eliminate the contribution of thisplasmid in the transfer, the PCR fragment obtained with oligonucleotides10 and 12 was cloned into another plasmid.

The erythromycin resistance gene was first amplified by PCR from plasmidpRClN and oligonucleotides 18 and 19, and then digested with therestriction enzymes BamHI and EcoRI. Using oligonucleotides 20 and 21, areplicative fragment of plasmid pPF107-3 (GenBank access number: Y12675)was amplified from the total DNA of the strain I-942 (deposited in theCNCM on Apr. 12, 1990) and then digested with the restriction enzymesBamHI and EcoRI. These two PCR fragments were ligated and used totransform MG1363. The novel plasmid obtained, called pLAB510, wasdigested with EcoRI and then ligated to the fragment obtained witholigonucleotides 10 and 12 by PCR amplification from plasmid pLAB500,and digested with EcoRI. This ligation was used to transform MG1363.This novel plasmid, pLAB511, was tested for its ability to betransferred from MG1363 to LM2301 (Table 1). It can be seen that thisnovel plasmid pLAB511 is indeed transferable between these two strainsby conjugation.

TABLE 1 Conjugative transfer efficiency Plasmid ORF Efficiency Relativeefficiency pLAB500 A,B,C,D,E 8.3 × 10⁻⁴ 100 pLAB501 B,C,D,E 7.1 × 10⁻⁴85 pLAB502 B,C,D,E 1.1 × 10⁻⁷ 0.01 pLAB503 E  <5 × 10⁻⁹ <0.0006 pLAB504C,D   1 × 10⁻⁷ 0.01 pLAB505 C,D 5.7 × 10⁻⁸ 0.007 pLAB506 B,C 5.7 × 10⁻⁸0.007 pLAB507 B,C,D 4.5 × 10⁻⁵ 5.4 pLAB508 B,C 4.2 × 10⁻⁷ 0.05 pLDP1 /9.2 × 10⁻⁸ 0.01 pLAB510 /  <5 × 10⁻⁹ <0.0006 pLAB511 B,C,D 1.3 × 10⁻³157

Efficiency=ratio of the number of transconjugants to the number ofreceptor cells.

Relative efficiency=ratio of the efficiency of conjugation for a givenplasmid to the efficiency of conjugation for pLAB500×100.

ORF =complete open reading frames contained in the plasmid.

EXAMPLE 5 Demonstration of the relaxation conferred by differentplasmids

Relaxation (formation of open circular forms of plasmids fromsupercoiled forms of plasmids) is involved in the transfer of certainplasmids (Plasmid-protein relaxation complexes in Staphylococcus aureus,R. Novick, J. Bacteriol. 1976; 127: 1177-1187). From comparisons withcomputerized data banks (GenBank), the protein deduced from ORF C showsa homology of 35.2% with Rlx, a relaxase from Staphylococcus aureus. Itcan therefore be assumed that this protein deduced from ORF C is also arelaxase. Plasmids pLAB500 to pLAB508, pLAB510, pLABS11 and pLDP1 weretherefore extracted and deposited on agarose gel. After migration, amongall these plasmids extracted from MG1363, only pLAB503, pLAB510 andpLDP1 do not have an open circular form, so ORF C is indeed involved inthe relaxation of plasmids.

EXAMPLE 6 Kinetics of pLAB500 transfer

Conjugations between MG1363 containing pLAB500 and LM2301 were performedat different times in order to evaluate the point at which transfer tookplace. It is found (cf. Table 2 below and FIG. 2) that conjugation hasalready started after 1 hour and that the peak of transfer efficiency isreached after about 4 hours. Conjugations every 10 minutes between 0 andone hour also showed that no conjugation took place before one hour.

TABLE 2 Kinetics of pLAB500 transfer Conjugation time (hours) Efficiencyof conjugation 0 <1.1 × 10⁻⁸   1 6.6 × 10⁻⁵ 2 9.8 × 10⁻⁵ 3 1.7 × 10⁻⁴ 43.4 × 10⁻⁴ 5 2.7 × 10⁻⁴ 6 2.2 × 10⁻⁴ 7 6.7 × 10⁻⁵ 8 4.2 × 10⁻⁵ 9 3.0 ×10⁻⁵ 29 7.9 × 10⁻⁵

Efficiency of conjugation=ratio of the number of transconjugants to thenumber of receptor cells.

23 1 5333 DNA Lactococcus lactis 1 gaattcaaaa atataatgct tattttagtattagtaacca tctctataat tttaaatata 60 ttaattaacc gatttattat ttttcatcacttagggataa tgaataatca aattaatatt 120 gatagtatat taagttcttt atcatgtttaggaaaaattt ttggtattgc cttattagcc 180 cccatactcg aagaaagtat tttcagagcgtctatttacc aaatcttcat taatgataaa 240 gtttcttttc ttatctctag cttactatttgcatttttac ataggggtta tagttgggtt 300 ttcttcacgt atctgccagt aagtttatgtatgacattta tctatcatcg cagaaaaata 360 ttgacagatt ccattctatt tcattcgttatttaatttat tagtattggg tttgaatttt 420 ttaatatgaa ataaatttta gaatagtacttactttttgg ataaatagta aaattataga 480 aacgattcat tattggttct cagatgtctatagagttgga cactttcagt gtttagataa 540 aaattaggac taacaagtaa ctactgaaatattaaccaat tatagtttat aaaaaaacga 600 aggataaata tacatgctag acattttaaataaagcaaga atacataaaa aatggttcct 660 attttcatat tcaattatct ctttttgtattacaattatt tatattgttt ttaatcacac 720 attttttaaa gttaattggg caaaatataatagcgatgac agctataaaa ataaagtaga 780 tgagatttta aagcatggag ttttctggattaatggaaat ttaacatcta ttagttcgcc 840 attattaatt tgccttttct tgcttggtgcattcttttca ttaactattt tcttcttaac 900 ttggagaaat ttatcgacta gaacatggaccccaattata tcctttcttg gatttctgat 960 tccatttatt catagtgatg gaaatttcataaatttattg attttatctt ttattcttat 1020 actatttggg gctatttcct ctgttcctagtcttagatat ttttaaatat tacagcccaa 1080 aatgaatact taaaaatatc attcactctttatatgctaa tacccttaag aagtctcaaa 1140 tacgaacgaa aaatattcta atagggtcatctatcacata aatctactct atgctaaaaa 1200 caaaaatctt atttaataat tatattctcatttctatctg tagtgtttat taatattttt 1260 gaaagataaa gatagaaaga attaatcattaaactatcag aaattacaaa aatggctagc 1320 atactgctta gccattttta ttttaattctgcgaaccgag ggggttaagg gtggagcttt 1380 gctccccctt acaagcgcca caatagccacgaagtggcta gcttgtgggt tgcttgccaa 1440 gactttatct ttattctagc ttttgagggtcgagcttgag cgtcggacac gaaaagtgct 1500 agaataaaga tatggacgga acgtccatggaaaggcgggg gttatgagcg aacacttaaa 1560 tatggctagc attaaaaaga aacaaccaaatcgaaaagaa cgaaaacaaa taagtttcag 1620 agtgagcgaa ccggaatatt taaaccttgagcgctcagcg aaagtcttaa atatttcggt 1680 gccggctttt gtcaaaaaga aggcacaaggcgctcgtgtc gtcgcaccta aaattaatcc 1740 agacgattca aaagaaatgg ctcgccagttggcagcactt ggcaataacg tgaatcaact 1800 cgctaaaagg gtcaatcaga ttgaatttgcggataaggac acgcaagagc gcctatcagc 1860 cgatttaagg cgcaccttac acggtctgggggaaatatgg cgacaactca cataaaacgc 1920 tcaaatggcg cttctagact cgtcaactacgctgaaaaaa gagcggttca aaaagacggc 1980 tataatttag acattgagta tgccaaatctgaactcaaac aagttcgaga aatttacgga 2040 aacaaagggg caacgcaagc ctacgcttcaagagtggcat tctctccgaa agaatttgac 2100 cctaaaaatg taaaagacca actaaaggcactagaaatcg ctaaagaaat ctattcaacc 2160 gcctatccca accaacaaat cgcaatgtatgttcacaacg acaccgattc cctccacgtt 2220 cacgccgtga ttggcgccat taacctactaacaggtaaaa aaatgcacgg caattggcaa 2280 gaataccgtg aaaggctcgt taaaataacggataaagtcg tggagaaaca tggcttaacc 2340 gtaaccgttc ctcatccgcg acctgaaaaaagaaccatgg cagaactaaa aatgaaagcc 2400 cgcggacaag tcacctggaa agacaaaatcagacaagccg tcgatacaac catgcgagaa 2460 gctcatatta gcgattttaa gagctttaaagagaaacttg gtgaactagc cgtcaatgtc 2520 attgaacgtg gcagagacct cacatatactctcacaggca ctgattataa atcacggggc 2580 gcaaaactcg gagaggatta caaaaaggagaccatttttt atgagctgga cagaagaaac 2640 caattacagt acggaacaag tcgacaacgacaaggtcgcg cttggcttga aggacgtgga 2700 gaacgccttg aacaagaaca acgcgctcgtcaaaaccttg caaaaagagc agaagaccta 2760 caaagaagaa ctctcgaaag cactgaacaatcaattcaac caagccatca acgacctcaa 2820 aaatcaaaag aaagaggact gggagggcctagcctctaat ttcgttaatc gcctcaatga 2880 cagcactaaa gaaatcacga atagccagcttgaaacggca caagaggaga tagacaagaa 2940 ctttgcacaa aaagaacaac gcttaaataaccttgttttt aacattgaga gcgccgaaca 3000 agccttaaat tttctaaata acagaatcaatcacatcaaa tcaaccgaaa gattgcagaa 3060 acaaaagcag ttctttcaag aaatcgtctttattttgggc gcaatcatgg caagcctcgg 3120 aacattactc ttggtctttg ccttttgttctgccctttat ggctttgggt ggcattccat 3180 ttggaactgg aaacaaatta ctccagtctatgatgctcca acctttcaaa aaggattatt 3240 tatcgtcatt aaagccattt taagtcttctattcttcatt tttggggtac tcgttatgtt 3300 tttccccttg gcgatttacc acaaattcctaaatagaatc aaaaatagtt acagaggttt 3360 tgggggatgg ctcaataaag tattcatcagaaagtaagac agtttttctg tctttttttt 3420 attgatcact tcgcaaaatt cgcgagcacaaaaattaaag ataatgcaaa ttaaaaattt 3480 cgtgttgtga gccttggcga acttttccttttggcaacct cggagagtgg gggaattttt 3540 gcgaaagcaa aaagggggca aagccccttaaaatgctttt gggaaaaatc tatgattttt 3600 gtccttttta aacctctttt ttcagaaggggggaaattta aaaaatgagg ctgaaaaatc 3660 cgagggtctc ttttatattt cttttataaatcttttaaac ctcttttagg gggctgggaa 3720 acgttgatat cactagcgtg aagcgttggttacatacccc taaatagggt actacatacc 3780 cctaaatagg gtactacata cccctaaatagggtactaca tacccctaaa tagggcgaga 3840 aagtttataa ccccttttta gggtacttcatttttttata acccttattt agggtacttc 3900 atttttttat aacccttatt tagggtgacaaaaacccccg ttataaaggt gttttgcttt 3960 tataacccct ttttagggtg cctctataacccttatttag ggtagatatt ttatataaaa 4020 attgctataa tttttataac cctaaagggataaagaaagg aagtataatg gttcatgaaa 4080 tagtacaata tcacaacgat tttaacactgttccacttag aggatttaat gaacgagaac 4140 gtagaattgt aatggcatta cttcatcaagtaaaaaataa agatgtcgaa gtggttcaat 4200 tagactttga tactttgcgt ggattatctggttggaatga tactttagct aaatctgaaa 4260 attccaatgc taaatttaac cggtatcttgaaaacttgtc tgataaaatt atgacattac 4320 gaggaactct aagaagtgaa gatggtttgcaagtagttaa atttagtctc tttccaacat 4380 ttattattga tgggaaaaat actatgaccctaaaagttca aattaaccct acttttaaat 4440 atcttactaa tatctttgat atgttcacagcttttgaatt agatgattat aatcgtatga 4500 acactagcta tgggcaagaa ctttatagattattaaaaca gtatcgaaca tctggttttt 4560 atcgtgtgaa gatagaggac ttgcgacatctattatcagt tcctgaaagc tataccaatg 4620 caaaaatgga tcaaaaagta ttttcaaaaactactgtaac tgaccttacc aatgcttttc 4680 cgaattttaa aatcaaacaa gaacgaggcactggtcgagg tcgaccaata attggttaca 4740 ccttcacttt cgataaagaa gccccaaataagtatgagct agaccgcaaa aagcaagaac 4800 aaattgccca attttggaaa tcaaatgaccctgagccaat gcctaatgca gttgctcaaa 4860 cggaatatca aaatcctgaa ttacgaaaagaaaaagaaga gctcgaaaaa cataacgcta 4920 gttttggaga cttattaaag ggctggttcaaaaaatagat aaatatgaaa tttaaaaaga 4980 aaaattatac tcctcaagta gatgaaaaagactgtggttg tgcggcatta tcaatgattt 5040 taaaaactta cgaaacagaa aagtcacttgcttcattttt attgaatcag aggataaaaa 5100 tgcataaagt atttgaaaaa attattacaattttttttgc ctttttttta tttttcattt 5160 ctcaaatccc aatatactac gtaaattataaaaataaaga aaataattta tatggaatat 5220 caaataaaat atcattacct tttatatttattgctttatt tgttattata atagcagttg 5280 ctctaggtaa aaaaagagga ttttaccatcattcgaagaa aacattagaa ttc 5333 2 2590 DNA Lactococcus lactis 2cataaatcta ctctatgcta aaaacaaaaa tcttatttaa taattatatt ctcatttcta 60tctgtagtgt ttattaatat ttttgaaaga taaagataga aagaattaat cattaaacta 120tcagaaatta caaaaatggc tagcatactg cttagccatt tttattttaa ttctgcgaac 180cgagggggtt aagggtggag ctttgctccc ccttacaagc gccacaatag ccacgaagtg 240gctagcttgt gggttgcttg ccaagacttt atctttattc tagcttttga gggtcgagct 300tgagcgtcgg acacgaaaag tgctagaata aagatatgga cggaacgtcc atggaaaggc 360gggggttatg agcgaacact taaatatggc tagcattaaa aagaaacaac caaatcgaaa 420agaacgaaaa caaataagtt tcagagtgag cgaaccggaa tatttaaacc ttgagcgctc 480agcgaaagtc ttaaatattt cggtgccggc ttttgtcaaa aagaaggcac aaggcgctcg 540tgtcgtcgca cctaaaatta atccagacga ttcaaaagaa atggctcgcc agttggcagc 600acttggcaat aacgtgaatc aactcgctaa aagggtcaat cagattgaat ttgcggataa 660ggacacgcaa gagcgcctat cagccgattt aaggcgcacc ttacacggtc tgggggaaat 720atggcgacaa ctcacataaa acgctcaaat ggcgcttcta gactcgtcaa ctacgctgaa 780aaaagagcgg ttcaaaaaga cggctataat ttagacattg agtatgccaa atctgaactc 840aaacaagttc gagaaattta cggaaacaaa ggggcaacgc aagcctacgc ttcaagagtg 900gcattctctc cgaaagaatt tgaccctaaa aatgtaaaag accaactaaa ggcactagaa 960atcgctaaag aaatctattc aaccgcctat cccaaccaac aaatcgcaat gtatgttcac 1020aacgacaccg attccctcca cgttcacgcc gtgattggcg ccattaacct actaacaggt 1080aaaaaaatgc acggcaattg gcaagaatac cgtgaaaggc tcgttaaaat aacggataaa 1140gtcgtggaga aacatggctt aaccgtaacc gttcctcatc cgcgacctga aaaaagaacc 1200atggcagaac taaaaatgaa agcccgcgga caagtcacct ggaaagacaa aatcagacaa 1260gccgtcgata caaccatgcg agaagctcat attagcgatt ttaagagctt taaagagaaa 1320cttggtgaac tagccgtcaa tgtcattgaa cgtggcagag acctcacata tactctcaca 1380ggcactgatt ataaatcacg gggcgcaaaa ctcggagagg attacaaaaa ggagaccatt 1440ttttatgagc tggacagaag aaaccaatta cagtacggaa caagtcgaca acgacaaggt 1500cgcgcttggc ttgaaggacg tggagaacgc cttgaacaag aacaacgcgc tcgtcaaaac 1560cttgcaaaaa gagcagaaga cctacaaaga agaactctcg aaagcactga acaatcaatt 1620caaccaagcc atcaacgacc tcaaaaatca aaagaaagag gactgggagg gcctagcctc 1680taatttcgtt aatcgcctca atgacagcac taaagaaatc acgaatagcc agcttgaaac 1740ggcacaagag gagatagaca agaactttgc acaaaaagaa caacgcttaa ataaccttgt 1800ttttaacatt gagagcgccg aacaagcctt aaattttcta aataacagaa tcaatcacat 1860caaatcaacc gaaagattgc agaaacaaaa gcagttcttt caagaaatcg tctttatttt 1920gggcgcaatc atggcaagcc tcggaacatt actcttggtc tttgcctttt gttctgccct 1980ttatggcttt gggtggcatt ccatttggaa ctggaaacaa attactccag tctatgatgc 2040tccaaccttt caaaaaggat tatttatcgt cattaaagcc attttaagtc ttctattctt 2100catttttggg gtactcgtta tgtttttccc cttggcgatt taccacaaat tcctaaatag 2160aatcaaaaat agttacagag gttttggggg atggctcaat aaagtattca tcagaaagta 2220agacagtttt tctgtctttt ttttattgat cacttcgcaa aattcgcgag cacaaaaatt 2280aaagataatg caaattaaaa atttcgtgtt gtgagccttg gcgaactttt ccttttggca 2340acctcggaga gtgggggaat ttttgcgaaa gcaaaaaggg ggcaaagccc cttaaaatgc 2400ttttgggaaa aatctatgat ttttgtcctt tttaaacctc ttttttcaga aggggggaaa 2460tttaaaaaat gaggctgaaa aatccgaggg tctcttttat atttctttta taaatctttt 2520aaacctcttt tagggggctg ggaaacgttg atatcactag cgtgaagcgt tggttacata 2580cccctaaata 2590 3 31 DNA Artificial sequence Description of theartificial sequenceoligonucleotide 3 tacatacgcg tctcatatat actttagatt g31 4 31 DNA Artificial sequence Description of the artificialsequenceoligonucleotide 4 tacatacgcg tgacttagaa gcaaacttaa g 31 5 31 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 5 ttaaatgatc agagctccac cgcggtggcg g 31 6 31 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 6 tattttgatc agaacaaaag ctgggtaccg g 31 7 31 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 7 atttatgatc atttccagtc gggaaacctg t 31 8 31 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 8 aattttgatc aagtatacct aataatttat c 31 9 31 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 9 tatgtgaatt cgacttagaa gcaaacttaa g 31 10 31DNA Artificial sequence Description of the artificialsequenceoligonucleotide 10 tatgtgaatt cagtatacct aataatttat c 31 11 29DNA Artificial sequence Description of the artificialsequenceoligonucleotide 11 tataagaatt ccgctcgtgt cgtcgcacc 29 12 29 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 12 tataagaatt ccataaatct actctatgc 29 13 30 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 13 tataagaatt cgtagatgag attttaaagc 30 14 29 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 14 tattagaatt ctatttaggg gtatgtaac 29 15 32 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 15 tattagaatt ccgtgatttc tttagtgctg tc 32 16 31DNA Artificial sequence Description of the artificialsequenceoligonucleotide 16 tatataagct tagtatacct aataatttat c 31 17 29DNA Artificial sequence Description of the artificialsequenceoligonucleotide 17 tatataagct tcacttcgca aaattcgcg 29 18 29 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 18 tatataagct tgagggtcga gcttgagcg 29 19 31 DNAArtificial sequence Description of the artificialsequenceoligonucleotide 19 tatataagct tcactcttta tatgctaata c 31 20 20DNA Artificial sequence Description of the artificialsequenceoligonucleotide 20 aggtttcccg actggaaatg 20 21 31 DNA Artificialsequence Description of the artificial sequenceoligonucleotide 21tacgtgaatt cagttttaaa tcaatctaaa g 31 22 31 DNA Artificial sequenceDescription of the artificial sequenceoligonucleotide 22 tacgtggatccatcggcata atcgttaaaa c 31 23 31 DNA Artificial sequence Description ofthe artificial sequenceoligonucleotide 23 tacgtgaatt cagaagaacccttaactaaa c 31

What is claimed is:
 1. An isolated nucleic acid sequence capable ofbeing transferred by conjugation, which comprises the sequence SEQ IDNo:2, or its complementary strand.
 2. The isolated nucleic acid sequenceaccording to claim 1 which is selected from the group consisting of: a)the nucleotide sequence of 5333 bp (SEQ ID No:1) or its complementarystrand; and b) any sequence hybridizing with the sequence a) understrict conditions.
 3. The isolated nucleic acid sequence according toclaim 1 which is selected from the group consisting of: a) thenucleotide sequence of 2590 bp (SEQ ID No:2) or its complementarystrand; and b) any sequence hybridizing with the sequence a) understrict conditions.
 4. A plasmid construct comprising a sequenceaccording to claim
 1. 5. A plasmid construct formed by incorporating asequence of claim 1 into plasmid pLDP1 or plasmid pLAB510.
 6. Abacterium which contains a sequence according to claim
 1. 7. Thebacterium according to claim 6 which is a lactic acid bacterium.
 8. Thelactic acid bacterium according to claim 7 which belongs to the speciesLactococcus lactis.
 9. A method of transferring genetic material to astrain of industrial interest, which comprises transferring the geneticmaterial by conjugation from a bacterium according to claim 6 to saidstrain of industrial interest.
 10. A strain of industrial interest intowhich genetic material has been transferred by the method of claim 9.11. The strain of industrial interest according to claim 10 which is L.lactis ssp lactis or L. lactis ssp cremoris.
 12. A plasmid constructcomprising a sequence according to claim
 2. 13. A plasmid constructcomprising a sequence according to claim
 3. 14. A bacterium whichcontains a sequence according to claim
 2. 15. The bacterium according toclaim 14 which is a lactic acid bacterium.
 16. The lactic acid bacteriumaccording to claim 15 which belongs to the species Lactococcus lactis.17. A bacterium which contains a sequence according to claim
 3. 18. Thebacterium according to claim 17 which is a lactic acid bacterium. 19.The lactic acid bacterium according to claim 18 which belongs to thespecies Lactococcus lactis.
 20. A bacterium which contains a plasmidconstruct according to claim
 4. 21. The bacterium according to claim 20which is a lactic acid bacterium.
 22. The lactic acid bacteriumaccording to claim 21 which belongs to the species Lactococcus lactis.23. A bacterium which contains a plasmid construct according to claim 5.24. The bacterium according to claim 23 which is a lactic acidbacterium.
 25. The lactic acid bacterium according to claim 24 whichbelongs to the species Lactococcus lactis.
 26. A bacterium whichcontains a plasmid construct according to claim
 12. 27. The bacteriumaccording to claim 26 which is a lactic acid bacterium.
 28. The lacticacid bacterium according to claim 27 which belongs to the speciesLactococcus lactis.
 29. A bacterium which contains a plasmid constructaccording to claim
 13. 30. The bacterium according to claim 29 which isa lactic acid bacterium.
 31. The lactic acid bacterium according toclaim 30 which belongs to the species Lactococcus lactis.
 32. A methodof transferring genetic material to a strain of industrial interest,which comprises transferring the genetic material by conjugation from abacterium according to claim 14 to said strain of industrial interest.33. A strain of industrial interest into which genetic material has beentransferred by the method of claim
 32. 34. The strain of industrialinterest according to claim 33 which is L. lactis ssp lactis or L.lactis ssp cremoris.
 35. A method of transferring genetic material to astrain of industrial interest, which comprises transferring the geneticmaterial by conjugation from a bacterium according to claim 17 to saidstrain of industrial interest.
 36. A strain of industrial interest intowhich genetic material has been transferred by the method of claim 35.37. The strain of industrial interest according to claim 36 which is L.lactis ssp lactis or L. lactis ssp cremoris.
 38. A method oftransferring genetic material to a strain of industrial interest, whichcomprises transferring the genetic material by conjugation from abacterium according to claim 20 to said strain of industrial interest.39. A strain of industrial interest into which genetic material has beentransferred by the method of claim
 38. 40. The strain of industrialinterest according to claim 39 which is L. lactis ssp lactis or L.lactis ssp cremoris.
 41. A method of transferring genetic material to astrain of industrial interest, which comprises transferring the geneticmaterial by conjugation from a bacterium according to claim 26 to saidstrain of industrial interest.
 42. A strain of industrial interest intowhich genetic material has been transferred by the method of claim 41.43. The strain of industrial interest according to claim 42 which is L.lactis ssp lactis or L. lactis ssp cremoris.
 44. A method oftransferring genetic material to a strain of industrial interest, whichcomprises transferring the genetic material by conjugation from abacterium according to claim 29 to said strain of industrial interest.45. A strain of industrial interest into which genetic material has beentransferred by the method of claim
 53. 46. The strain of industrialinterest according to claim 45 which is L. lactis ssp lactis or L.lactis ssp cremoris.